FIG. 1.

Effect of kinase signaling and As₂O₃ on the interaction between T3Rα and SMRT. (A) Effect of hormone on SMRT interaction with nuclear hormone receptors in a mammalian two-hybrid assay. A pSG5 plasmid containing either GAL4DBD alone (empty DBD), a GAL4DBD fused with the receptor interaction domain of SMRT (amino acids 751 to 1495; DBD-SMRT), or a GAL4DBD fused with a region of SMRT lacking the receptor interaction domain (amino acids 751 to 1074, DBD-SMRT ΔRID) were cotransfected into CV-1 cells with a GAL4(17-mer) thymidine kinase promoter-luciferase reporter and a pSG5-GAL4AD construct by a calcium phosphate precipitation method. Cotransfected GAL4AD constructs are indicated below the panels (empty, GAL4AD domain alone; T3R, GAL4-AD fused with T3Rα ligand-binding domain; P156R, GAL4-AD fused with a T3Rα construct with a mutation that disrupts SMRT association; T3R-vA, GAL4-AD fused with the analogous region of the v-Erb A mutant form of T3Rα, v-Erb A; VDR, GAL4-AD fused with a vitamin D₃ receptor ligand-binding domain). The cells were incubated in the absence or presence of 1 μM cognate hormone (T3), the cells were harvested, and luciferase activity was determined relative to β-galactosidase activity of pCH110 plasmid introduced as an internal control. Data are averages and standard deviations from duplicate experiments. (B) Inhibition of two-hybrid SMRT-T3Rα interaction by v-Erb B, MEKK-1, and MEK-1. A pSG5 plasmid expressing a GAL4DBD alone (empty DBD) or a GAL4DBD fused with the receptor interaction domain of SMRT (DBD-SMRT) was transiently cotransfected into CV-1 cells by Lipofectin, with either an empty pSG5-GAL4AD vector, the pSG5-GAL4AD fused to the ligand-binding domain of T3Rα, or T3R-vA. In addition, the cells were cotransfected with 100 ng of expression plasmid for either v-Erb B, v-Ras, a full-length MEKK-1, or a MEK-1 clone, as indicated at the bottom. The cells were incubated in the absence of T3, and the luciferase activity was determined and normalized to β-galactosidase activity for each sample. (C) Inhibition of the mammalian two-hybrid interaction between SMRT and T3Rα by As₂O₃. The protocol for panel B was repeated, but in the absence or presence or absence of 20 μM As₂O₃, as indicated at the bottom. A control two-hybrid interaction between a DBD-SMRT construct and an AD-T3Rα construct was assayed under the same conditions to detect any nonspecific effects of arsenite on the two-hybrid system (see the text). The scale for the v-Erb A interaction with SMRT (right axis) differs from that for the other three panels (left axis).